Thrombin is an important serine protease in hemostasis and thrombosis. One of the key actions of thrombin is cellular modulation via receptor activation. A functional human thrombin receptor (PAR-1), cloned by Coughlin in 1991 (T. -K. Vu, Cell 1991, 64, 1057), was found to be a member of the G-protein coupled receptor (GPCR) superfamily. The receptor activation putatively occurs by N-terminal recognition and proteolytic cleavage at the Arg-41/Ser-42 peptide bond to reveal a truncated N-terminus. This new N-terminus acting as a tethered ligand to recognize a site on the receptor, can trigger activation and signal transduction leading to platelet aggregation. Since 1991, three other protease-activated receptors with extensive homology to the thrombin receptor, xe2x80x9cPAR-2xe2x80x9d (S. Nystedt, Proc. Natl. Acad. Sci USA 1994, 91, 9208), xe2x80x9cPAR-3xe2x80x9d (H. Ishihara, Nature 1997, 386, 502), and xe2x80x9cPAR-4xe2x80x9d (W.-F. Xu, Proc. Natl. Acad. Sci USA 1998, 95, 6642), have been cloned. Thrombin receptor (PAR-1) specific antibody-induced blockade of the platelet thrombin receptor has shown efficacy against arterial thrombosis in vivo (J. J. Cook Circulation 1995, 91, 2961). Hence, antagonists of the thrombin receptor (PAR-1) are useful to block these protease-activated receptors and, as such, may be used to treat platelet mediated thrombotic disorders such as myocardial infarction, stroke, restenosis, angina, atherosclerosis, and ischemic conditions.
The thrombin receptor (PAR-1) has also been identified on other cell types: endothelial, fibroblast, renal, osteosarcoma, smooth muscle, myocytes, tumor, and neuronal/glia. Thrombin activation of endothelial cells upregulates P-selectin to induce polymorphonuclear leukocyte adhesionxe2x80x94an inflammatory response of the vessel wall (Y. Sugama, J. Cell Biol. 1992, 119, 935). In fibroblasts, thrombin receptor (PAR-1) activation induces proliferation and transmission of mitogenic signals (D. T. Hung, J. Cell Biol. 1992, 116, 827). Thrombin has been implicated in osteoblast proliferation through its activation of osteoblast cells (D. N. Tatakis, Biochem. Biophys. Res. Commun. 1991, 174, 181). Thrombin has been implicated in the regulation and retraction of neurons (K. Jalink, J. Cell. Biol. 1992, 118, 411). Therefore, in this context, the antagonist compounds of this invention may also be useful against inflammation, osteoporosis, Angiogenesis related disorders, cancer, neurodegenerative disorders, hypertension, heart failure, arrhythmia, glomerulonephritis.
The compounds of the present invention are a structurally novel class of benzimidazolone peptidomimetics represented by the general formula (I) below.
The present invention is directed to structurally novel compounds represented by the following general formula (I): 
wherein
A1 and A2 are each independently a D- or L-amino acid selected from the group consisting of alanine, xcex2-alanine, arginine, homoarginine, cyclohexylalanine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)xe2x80x94), 2,3 diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)xe2x80x94), glutamine, glycine, indanylglycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)xe2x80x94), valine, methionine, proline, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), tetrahydroisoquinoline-3-COOH, threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)xe2x80x94), and an unsubstituted or substituted aromatic amino acid selected from the group consisting of phenylalanine, heteroarylalanine, naphthylalanine, homophenylalanine, histidine, tryptophan, tyrosine, arylglycine, heteroarylglycine, aryl-xcex2-alanine, and heteroaryl-xcex2-alanine wherein the substituents on the aromatic amino acid are independently selected from one or more of halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;
Preferably, A1 and A2 are each independently an L-amino acid selected from the group consisting of alanine, xcex2-alanine, arginine, homoarginine, cyclohexylalanine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)xe2x80x94), 2,3 diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)xe2x80x94), glutamine, glycine, indanylglycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)xe2x80x94), valine, methionine, proline, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), tetrahydroisoquinoline-3-COOH, threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)xe2x80x94), and an unsubstituted or substituted aromatic amino acid selected from the group consisting of phenylalanine, heteroarylalanine, naphthylalanine, homophenylalanine, histidine, tryptophan, tyrosine, arylglycine, heteroarylglycine, aryl-xcex2-alanine, and heteroaryl-xcex2-alanine wherein the substituents on the aromatic amino acid are independently selected from one or more of halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;
R1 is selected from amino, C1-C8 alkylamino, C1-C8 dialkylamino, arylamino, arC1-C8 alkylamino, C3-C8 cycloalkylamino, heteroalkylC1-C8 alkylamino, heteroalkylC1-C8 alkyl-N-methylamino, C1-C8 dialkylaminoC1-C8 alkylamino, xe2x80x94N(C1-C8alkyl)xe2x80x94C1-C8 alkyl-N(C1-C8alkyl)2, N(C1-C8 alkyl)(C1-C8 alkenyl), xe2x80x94N(C1-C8alkyl)(C3-C8cycloalkyl), heteroalkyl or substituted heteroalkyl wherein the substituent on the heteroalkyl is selected from oxo, amino, C1-C8 alkoxyC1-C8 alkyl, C1-C8 alkylamino or C1-C8 dialkylamino;
Preferably, R1 is selected from amino, C1-C6 alkylamino, C1-C6 dialkylamino, arylamino, arC1-C6 alkylamino, heteroalkylC1-C6 alkylamino, xe2x80x94N(C1-C6alkyl)xe2x80x94C1-C6alkyl-N(C1-C6alkyl)2, heteroalkyl or substituted heteroalkyl wherein the substituent on the heteroalkyl is selected from oxo, amino, C1-C6alkoxyC1-C6 alkyl, C1-C6 alkylamino or C1-C6 dialkylamino;
R2 and R3 are each independently selected from hydrogen, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C8 alkyl, aryl, heteroalkyl, substituted heteroalkyl (wherein the substituent on the heteroalkyl is one or more substituents independently selected from C1-C8 alkoxycarbonyl, C1-C8 alkyl, or C1-C4 alkylcarbonyl), heteroalkylC1-C8 alkyl, indanyl, acetamidinoC1-C8 alkyl, aminoC1-C8 alkyl, C1-C8 alkylaminoC1-C8 alkyl, C1-C8, dialkylaminoC1-C8 alkyl, unsubstituted or substituted heteroarylC1-C8 alkyl or unsubstituted or substituted arC1-C8 alkyl, wherein the substituent on the aralkyl or heteroarylalkyl group is one or more substituents independently selected from halogen, nitro, amino, C1-C8 alkyl, C1-C8 alkoxy, hydroxy, cyano, C1-C4 alkylcarbonyl, C1-C8 alkoxycarbonyl, hydroxyC1-C8 alkyl or aminosulfonyl; or
R2 and R3 together with the nitrogen to which they are attached, alternatively form an unsubstituted or substituted heteroalkyl group selected from piperidinyl, piperazinyl, morpholinyl or pyrrolidinyl, wherein the substituent is one or more substituents independently selected from C1-C8 alkyl C1-C8 alkoxycarbonyl or C1-C4 alkylcarbonyl;
Preferably, R2 is selected from hydrogen or C1-C6 alkyl;
R3 is selected from C1-C8 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkylC1-C6 alkyl, aryl, heteroarylC1-C6 alkyl, substituted heteroarylC1-C6 alkyl wherein the substituent is C1-C4 alkyl, heteroalkyl, heteroalkylC1-C6 alkyl, indanyl, acetamidinoC1-C6 alkyl, aminoC1-C6 alkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 dialkylaminoC1-C6 alkyl, arC1-C8 alkyl, substituted arC1-C8 alkyl wherein the substituent on the aralkyl group is one to five substituents independently selected from halogen, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, hydroxyalkyl or aminosulfonyl; or
R2 and R3, together with the nitrogen to which they are attached, alternatively form an unsubstituted or substituted heteroalkyl group selected from piperidinyl, piperazinyl or pyrrolidinyl, wherein the substituent is independently one or two substituents selected from C1-C6 alkyl;
R4 is selected from unsubstituted or substituted aryl, arC1-C8 alkyl, C3-C8 cycloalkyl, or heteroaryl, where the substituents on the aryl, arC1-C8 alkyl, cycloalkyl or heteroaryl group are independently selected from one or more of halogen, nitro, amino, cyano, hydroxyalkyl, C1-C8 alkyl, C1-C8 alkoxy, hydroxy, C1-C4 alkylcarbonyl, C1-C8 alkoxycarbonyl, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy or C1-C4 alkylsulfonyl;
Preferably, R4 is selected from unsubstituted or substituted aryl, arC1-C6 alkyl, C3-C6 cycloalkyl or heteroaryl, where the substituents on the aryl, aralkyl, cycloalkyl or heteroaryl group are independently selected from one to three substituents selected from halogen, cyano, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkoxycarbonyl, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy or C1-C4 alkylsulfonyl;
R5 is selected from hydrogen or C1-C8 alkyl; preferably, R5 is hydrogen;
X is oxygen or sulfur; preferably, X is oxygen;
m is an integer selected from 0, 1, 2 or 3;
n is an integer selected from 2 or 3; and
p is an integer selected from 0 or 1; preferably, p is 1;
and pharmaceutically acceptable salts thereof.
In a preferred embodiment of the present invention:
A1 is an L-amino acid selected from the group consisting of alanine, arginine, cyclohexylalanine, glycine, proline, tetrahydroisoquinoline-3-COOH, and an unsubstituted or substituted aromatic amino acid selected from the group consisting of phenylalanine, naphthylalanine, homophenylalanine, and O-methyl tyrosine, wherein the substituents on the aromatic amino acid are independently selected from one to five of (preferably, one to three of) halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;
A2 is an L-amino acid selected from the group consisting of alanine, xcex2-alanine, arginine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)xe2x80x94), 2,3-diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)xe2x80x94), glutamine, glycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)xe2x80x94), valine, methionine, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)xe2x80x94), and an unsubstituted or substituted aromatic amino acid selected from the group consisting of phenylalanine, heteroarylalanine, and histidine, wherein the substituents on the aromatic amino acid are independently selected from one to five of (preferably, one to three of) halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;
R2 is selected from hydrogen or C1-C4 alkyl;
m is 1 and n is 2;
and all other variables are as defined previously;
and pharmaceutically acceptable salts thereof.
In a class of the invention:
A1 is an L-amino acid selected from the group consisting of alanine, arginine, cyclohexylalanine, glycine, proline, and an unsubstituted or substituted aromatic amino acid selected from the group consisting of phenylalanine, naphthylalanine, homophenylalanine, and O-methyl tyrosine, wherein the substituents on the aromatic amino acid are independently one to two substituents selected from halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;
A2 is an L-amino acid selected from the group consisting of alanine, xcex2-alanine, arginine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)xe2x80x94), 2,3-diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)xe2x80x94), glutamine, glycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)xe2x80x94), valine, methionine, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)xe2x80x94), and an unsubstituted or substituted aromatic amino acid selected from the group consisting of phenylalanine, heteroarylalanine, and histidine, wherein the substituents on the aromatic amino acid are independently one to two substituents selected from halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;
R1 is selected from dimethylamino, diethylamino, di-(n-propyl)amino, 
Preferably, R1 is: 
R2 is selected from hydrogen, methyl or ethyl;
R3 is selected from 2-indanyl, phenyl, cyclohexylmethyl, cyclopentyl, pyridylmethyl, furanylmethyl, 2-(4-methylfuranyl)methyl, thienylmethyl, diphenylmethyl, 4-imidazolylethyl, 2-(4-N-methyl)imidazolylethyl, n-octyl, phenyl-n-propyl, aminoethyl, aminopropyl, amino-n-pentyl, dimethylaminoethyl, 4-aminophenylsulfonylaminomethyl, acetamidinylethyl, 2-N-pyrrolidinylethyl, N-ethoxycarbonylpiperidinyl, unsubstituted or substituted phenylethyl or unsubstituted or substituted benzyl wherein the substituents on the phenylethyl or benzyl are independently one or two substituents selected from methyl, fluorine, chlorine, nitro, methoxy, methoxycarbonyl or hydroxymethyl; or
R2 and R3, together with the nitrogen to which they are attached, alternatively form a heteroalkyl group selected from piperidinyl, or 4-(N-methyl)piperazinyl;
R4 is selected from cyclohexyl, 2-naphthyl, phenylethyl, 4-fluorophenylethyl, or unsubstituted or substituted phenyl, where the substituents on the phenyl are independently selected from one to two substituents selected from fluorine, chlorine, iodine, methyl, cyano, or trifluoromethyl;
Preferably, R4 is 2,6-dichlorophenyl or 2-methylphenyl;
and all other variables are as defined previously;
and pharmaceutically acceptable salts thereof.
In a subclass of the invention,
A1 is selected from 3,4-Difluorophenylalanine or 4-Chlorophenylalanine;
A2 is selected from 2,4-Diaminobutyric acid or 4-Pyridylalanine;
R2 is hydrogen;
R3 is selected from benzyl or 2-aminoethyl;
and all other variables are as defined previously;
and pharmaceutically acceptable salts thereof.
Illustrative of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any of the compounds described above. Illustrating the invention is a pharmaceutical composition made by mixing any of the compounds described above and a pharmaceutically acceptable carrier. An illustration of the invention is a process for making a pharmaceutical composition comprising mixing any of the compounds described above and a pharmaceutically acceptable carrier.
An example of the invention is a method of treating a disorder (preferably, a platelet-mediated thrombotic disorder) selected from arterial and/or venous thrombosis, acute myocardial infarction, reocclusion following thrombolytic therapy and/or angioplasty, inflammation, unstable angina, stroke, restenosis, atherosclerosis, ischemic conditions, hypertension, heart failure, arrhythmia, glomerulonephritis, osteoporosis, Angiogenesis related disorders, cancer, neurodegenerative disorders and a variety of vaso-occlusive disorders in a subject in need thereof comprising administering to the subject a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above. In a preferred embodiment, the therapeutically effective amount of the compound is from about 0.1 mg/kg/day to about 300 mg/kg/day.
Also included in the invention is the use of any of the compounds described above for the preparation of a medicament for a disorder (preferably, a platelet-mediated thrombotic disorder) selected from arterial and/or venous thrombosis, acute myocardial infarction, reocclusion following thrombolytic therapy and/or angioplasty, inflammation, unstable angina, stroke, restenosis, atherosclerosis, ischemic conditions, hypertension, heart failure, arrhythmia, glomerulonephritis, osteoporosis, Angiogenesis related disorders, cancer, neurodegenerative disorders or a variety of vaso-occlusive disorders in a subject in need thereof.
More particularly, the present invention is directed to compounds of the following formula (I): 
wherein A1, A2, R1, R2, R3, R4, R5, X, m, n and p are as previously defined. In a particularly preferred embodiment, the compounds have the formula 
The compounds of the present invention are thrombin receptor antagonists and as such are useful in treating thrombosis, restenosis, hypertension, heart failure, arrhythmia, myocardial infarction, glomerulonephritis, reocclusion following thrombolytic therapy, reocclusion following angioplasty, inflammation, angina, stroke, atherosclerosis, ischemic conditions, a vaso-occlusive disorder, neurodegenerative disorders, Angiogenesis related disorders and cancer. These compounds are also useful as antithrombotics in conjunction with fibrinolytic therapy (e.g., t-PA or streptokinase).
In the compounds of formula (I), the amino acid residues comprising the A1 and A2 substituents are attached to the adjacent moiety according to standard nomenclature so that the amino-terminus (N-terminus) of the amino acid is drawn on the left and the carboxy-terminus of the amino acid is drawn on the right. So, for example, in Compound 1 in Table 1, where A1 is 3,4-difluorophenylalanine and A2 is Dbu (2,4-Diaminobutyric acid), the N-terminus of the 3,4-difluorophenylalanine (A1) is attached to the carbonylgroup and the carboxy-terminus of the 3,4-difluorophenylalanine (A1) is attached to the N-terminus of the A2 substituent (Dbu), similarly, the N-terminus of the Dbu (A2) is attached to the carboxy-terminus of the A1 substituent and the carboxy-terminus of the Dbu (A2) is attached to the Nxe2x80x94R2R3 group.
When a particular group is xe2x80x9csubstitutedxe2x80x9d (e.g., Phe, aryl, heteroalkyl, heteroaryl), that group may have one or more substituents, preferably from one to five substituents, more preferably from one to three substituents, most preferably from one to two substituents, independently selected from the list of substituents.
Under standard nomenclature used throughout this disclosure, the terminal portion of the designated side chain is described first, followed by the adjacent functionality toward the point of attachment. Thus, for example, a xe2x80x9cphenylC1-C6 alkylamidoC1-C6alkylxe2x80x9d substituent refers to a group of the formula 
The compounds of the present invention may also be present in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salt generally takes a form in which the basic nitrogen is protonated with an inorganic or organic acid. Representative organic or inorganic acids include hydrochloric, hydrobromic, hydriodic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic, mandelic, methanesulfonic, hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, salicylic, saccharinic or trifluoroacetic.
Where the compounds according to this invention have at least one chiral center, they may accordingly exist as enantiomers. Where the compounds possess two or more chiral centers, they may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention. Furthermore, some of the crystalline forms for the compounds may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this invention.
The term xe2x80x9csubjectxe2x80x9d as used herein, refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
As used herein, unless otherwise noted alkyl and alkoxy whether used alone or as part of a substituent group, include straight and branched chains having 1 to 8 carbon atoms, or any number within this range. For example, alkyl radicals include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, 3-(2-methyl)butyl, 2-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-hexyl and 2-methylpentyl. Alkoxy radicals are oxygen ethers formed from the previously described straight or branched chain alkyl groups. Cycloalkyl groups contain 3 to 8 ring carbons and preferably 5 to 7 carbons. Similarly, alkenyl and alkynyl groups include straight and branched chain alkenes and alkynes having 1 to 8 carbon atoms, or any number within this range.
The term xe2x80x9carylxe2x80x9d as used herein refers to an unsubstituted or substituted aromatic group such as phenyl and naphthyl. The term xe2x80x9caroylxe2x80x9d refers to the group xe2x80x94C(O)-aryl.
The term xe2x80x9cheteroalkylxe2x80x9d as used herein represents an unsubstituted or substituted stable three to seven membered monocyclic saturated ring system which consists of carbon atoms and from one to three heteroatoms selected from N, O or S, and wherein the nitrogen or sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroalkyl group may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heteroalkyl groups include, but are not limited to azetidinyl, piperidinyl, pyrrolidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl, oxoazepinyl, azepinyl, tetrahydrofuranyl, dioxolanyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydrooxazolyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone and oxadiazolyl. Preferred heteroalkyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, azetidinyl and tetrahydrothiazolyl.
The term xe2x80x9cheteroarylxe2x80x9d as used herein represents an unsubstituted or substituted stable five or six membered monocyclic aromatic ring system or an unsubstituted or substituted nine or ten membered benzo-fused heteroaromatic ring system or bicyclic heteroaromatic ring system which consists of carbon atoms and from one to four heteroatoms selected from N, O or S, and wherein the nitrogen or sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroaryl group may be attached at any heteroatom or carbon atom that results in the creation of a stable structure. Examples of heteroaryl groups include, but are not limited to pyridyl, pyridazinyl, thienyl, furanyl, imidazolyl, isoxazolyl, oxazolyl, pyrazolyl, pyrrolyl, thiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzofuranyl, benzothienyl, benzisoxazolyl, benzoxazolyl, benzopyrazolyl, indolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl adeninyl or quinolinyl. Prefered heteroaryl groups include pyridyl, pyrrolyl, pyrazinyl, thiadiazolyl, pyrazolyl, thienyl, triazolyl and quinolinyl.
The term xe2x80x9caralkylxe2x80x9d means an alkyl group substituted with one, two or three aryl groups (e.g., benzyl, phenylethyl, diphenylmethyl, triphenylmethyl). Similarly, the term xe2x80x9caralkoxyxe2x80x9d indicates an alkoxy group substituted with an aryl group (e.g., benzyloxy). The term aminoalkyl refers to an alkyl group substituted with an amino group (i.e., -alkyl-NH2). The term xe2x80x9calkylaminoxe2x80x9d refers to an amino group substituted with an alkyl group (i.e., xe2x80x94NH-alkyl). The term xe2x80x9cdialkylaminoxe2x80x9d refers to an amino group which is disubstituted with alkyl groups wherein the alkyl groups can be the same or different (i.e., xe2x80x94N-[alkyl]2).
The term xe2x80x9cacylxe2x80x9d as used herein means an organic radical having 1 to 6 carbon atoms (branched or straight chain) derived from an organic acid by removal of the hydroxyl group.
The term xe2x80x9coxoxe2x80x9d refers to the group xe2x95x90O.
The term xe2x80x9ccarbonylxe2x80x9d refers to the group C(O).
The term xe2x80x9chalogenxe2x80x9d shall include iodine, bromine, chlorine and fluorine.
The term xe2x80x9cN(CH2)4xe2x80x9d as used herein (e.g., in the Tables), refers to a pyrrolidinyl group having the structure 
Similarly, xe2x80x9cN(CH2)5xe2x80x9d, xe2x80x9cN(CH2)3xe2x80x9d, xe2x80x9cNCH2S(CH2)2xe2x80x9d and xe2x80x9cN(CH2)2O(CH2)2xe2x80x9d refer to 
respectively.
Similarly xe2x80x9cC6H11xe2x80x9d and xe2x80x9cC5H9xe2x80x9d (or xe2x80x9cc-C6H11xe2x80x9d and xe2x80x9cc-C5H9xe2x80x9d) refer to cyclohexyl and cyclopentyl groups, respectively.
Whenever the term xe2x80x9calkylxe2x80x9d or xe2x80x9carylxe2x80x9d or either of their prefix roots appear in a name of a substituent (e.g., aralkyl, dialkylamino) it shall be interpreted as including those limitations given above for xe2x80x9calkylxe2x80x9d and xe2x80x9caryl.xe2x80x9d Designated numbers of carbon atoms (e.g., C1-C6) shall refer independently to the number of carbon atoms in an alkyl or cycloalkyl moiety or to the alkyl portion of a larger substituent in which alkyl appears as its prefix root.
As used herein, the term xe2x80x9cphosgene equivalentxe2x80x9d represents the class of carbonic acid derivatives which include 4-nitrophenyl chloroformate, phosgene or xe2x80x9cCOCl2,xe2x80x9d phenyl chloroformate, triphosgene or xe2x80x9c(CCl3O)2CO,xe2x80x9d carbonyldiimidazole, diethyl carbonate or diphenyl carbonate.
It is intended that the definition of any substituent or variable at a particular location in a molecule be independent of its definitions elsewhere in that molecule. It is understood that substituents and substitution patterns on the compounds of this invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth herein.
As used herein, the term xe2x80x9ccompositionxe2x80x9d is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. Accordingly, pharmaceutical compositions containing the compounds of the present invention as the active ingredient as well as methods of preparing the instant compounds are also part of the present invention.
Particularly preferred compounds of the present invention and their biological data are shown in Tables 1 and 2, following; the amino acids bear the xe2x80x9cLxe2x80x9d absolute configuration unless denoted otherwise. The Tables contain IC50 values (xcexcM) of the compounds in a thrombin receptor binding assay, and IC50 values (xcexcM) against platelet aggregation stimulated by thrombin.
The antagonists of the present invention may be prepared via a convergent solution-phase synthesis by coupling an aminobenzimidazolone intermediate AAG4 with a dipeptide amine AAG6 via a urea linkage as described in the general Scheme AAGeneric. The appropriately nitro substituted benzimidazolone AAG1 (Scheme AAGeneric) was alkylated with a substituted aralkyl or heteroaryl alkyl halide and a base such as sodium hydride in a dipolar aprotic solvent such as DMF to give AAG2 as a mixture of two regioisomers. Two isomers were separated by silica gel column and then alkylated, with an aminoalkyl halide and a base such as sodium hydride in a dipolar aprotic solvent such as DMF to give two regioisomers AAG3, respectively. Reduction of nitro group in AAG3 in a classical manner with for example iron and acetic acid or with a newer method such as dimethyl hydrazine and iron to gave aminobenzimidazolone intermediate AAG4.
Dipeptide amine AAG6 can be synthesized from the corresponding protected amino acids using standard peptide coupling conditions. Thus, an Fmoc protected amino-acid (A2) AAG5 (Scheme AAGeneric) was coupled to amine R2R3NH using a coupling agent, such as dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT) in a dipolar aprotic solvent such as DMF to give the amide. The amide was then Fmoc deprotected with a dialkylamine in a dipolar aprotic solvent, such as diethylamine in acetonitrile. The resulting amine was coupled to the second Fmoc protected amino-acid (A1) in the same way with a coupling agent, such as DIC and HOBT in a dipolar aprotic solvent such as DMF to give the dipeptide. The dipeptide was then Fmoc deprotected as above with a dialkylamine in a dipolar aprotic solvent such as acetonitrile to afford dipeptide amine AAG6.
Aminobenzimidazolone intermediate AAG4 was treated with a phosgene equivalent such as 4-nitrophenyl chloroformate, phosgene or xe2x80x9cCOCl2,xe2x80x9d phenyl chloroformate, triphosgene or xe2x80x9c(CCl3O)2CO,xe2x80x9d carbonyldiimidazole, diethyl carbonate or diphenyl carbonate.and a base such as diisopropylethylamine in a solvent such as dichloromethane, and to this was then added dipeptide amine AAG6 to give a urea. Removal of the protecting group, if necessary, such as the Boc group with an acid such as trifluoroacetic acid from the side chain of dipeptide afforded final targets AAG7. 
As a typical example of this convergent solution-phase method, Compound 1 may be synthesized as described in Scheme AA. Thus, treatment of 5-nitrobenzimidazolone AAI with 2,6-diCl-Bn-Br in the presence of NaH in DMF afforded two regioisomers AA2a and AA3a (ca. 1:1 ratio), which were then separated by flash column. A small amount of bis-alkylated product was also obtained. The isolated AA3a was further alkylated with 2-chloroethylpyrrolidine by using NaH as a base in DMF to give the di-alkylated product, which was then subjected to nitro reduction with Me2NNH2/FeCl3 to provide aminobenzimidazolone intermediate AA4. Coupling of N-xcex1-Fmoc-N-xcex3-Boc-diaminobutyric acid (AA5) with benzyl amine in the presence of DCC and HOBt was followed by de-protection of the Fmoc group with diethylamine. The resulting intermediate was coupled with Fmoc-3,4-diF-Phe-OH using DIC/HOBt and treated with diethylamine to give dipeptide amine AA6. Urea formation between the dipeptide amine AA6 and aminobenzimidazolone intermediate AA4 in the presence of 4-nitrophenylchloroformate was followed by de-protection of the Boc group with TFA to afford the target compound 1.
In an alternative approach to the targets, the preferred intermediate AA3 could be prepared via a regioselective method. For example, reductive alkylation of 4-nitro-1,2-phenylenediamine AB1 with one equivalent of 4-fluorobenzaldehyde in the presence of sodium triacetoxyborohydride gave mono-alkylated product AB2 which was then cyclized with N,Nxe2x80x2-disuccinimidyl carbonate in acetonitrile to give AA3c (Scheme AB). By following the same procedure as described in Scheme M, the target 6 was obtained from intermediate AA3c. 
The side-chain amine in antagonists such as Compound 1 and Compound 8 may be converted to other functional groups such as acetamidine and guanidine by using standard procedures. For example, the acetamidine and guanidine groups can be introduced by treating the side-chain amine with S-2-naphthylmethyl thioacetimidate hydrobromide and 2-methyl-2-thiopseudourea, respectively.
The thioureidoindoles [when X is S, as in general formula (I)] may be prepared as described hereinafter. The aminobenzimidazolone substrate is reacted with thiocarbonyldiimidazole in a chlorinated solvent and then with the imidazole by-product filtered from the solution. The solution can then be concentrated to afford the N-imidazolyl-Nxe2x80x2-benzimidazolonyl-thioamide. This intermediate is then reacted with a peptide amine in a polar, aprotic solvent with heating (from about 80xc2x0 C. to about 100xc2x0 C.) to afford the N-peptido-Nxe2x80x2-benzimidazolonyl-thiourea product.
Amidobenzimidazolone targets [when p is 0 and X is O, as in general formula (I)] may be prepared from a dipeptide amine AAG6 (Scheme AAGeneric) and a benzimidazolone carboxylic acid intermediate by using standard coupling conditions such as DCC/HOBt. The required benzimidazolone carboxylic acid intermediates can be prepared by using the method as described for aminobenzimidazolone intermediate AAG4 in Scheme AAGeneric from the appropriate benzimidazolone carboxylic acid esters.
The utility of the compounds to treat PAR-1 mediated disorders (e.g., thrombotic disorders) can be determined according to the procedures described herein. The present invention therefore provides a method of treating PAR-1 mediated disorders (e.g., thrombotic disorders) in a subject in need thereof which comprises administering any of the compounds as defined herein in a quantity effective to treat PAR-1 mediated disorders. The compound may be administered to a patient by any conventional route of administration, including, but not limited to, intravenous, oral, subcutaneous, intramuscular, intradermal and parenteral.
The present invention also provides pharmaceutical compositions comprising one or more compounds of this invention in association with a pharmaceutically acceptable carrier.
To prepare the pharmaceutical compositions of this invention, one or more compounds of formula (I) or salt thereof of the invention as the active ingredient, is intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, which carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral such as intramuscular. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations, such as, for example, suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like; for solid oral preparations such as, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques. For parenterals, the carrier will usually comprise sterile water, though other ingredients, for example, for purposes such as aiding solubility or for preservation, may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. The pharmaceutical compositions herein will contain, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful and the like, an amount of the active ingredient necessary to deliver an effective dose as described above. The pharmaceutical compositions herein will contain, per unit dosage unit, e.g., tablet, capsule, powder, injection, suppository, teaspoonful and the like, from about 0.03 mg/kg to about 100 mg/kg (preferred from about 0.1 mg/kg to about 30 mg/kg) of a compound of the present invention and may be given at a dosage from about 0.1 mg/kg/day to about 300 mg/kg/day (preferred from about 1 mg/kg/day to about 50 mg/kg/day). The dosages, however, may be varied depending upon the requirement of the patients, the severity of the condition being treated and the compound being employed. The use of either daily administration or post-periodic dosing may be employed.
Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, autoinjector devices or suppositories for oral parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. Alternatively, the composition may be presented in a form suitable for once-weekly or once-monthly administration; for example, an insoluble salt of the active compound, such as the decanoate salt, may be adapted to provide a depot preparation for intramuscular injection. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from about 0.1 mg to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of material can be used for such enteric layers or coatings, such materials including a number of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavoured syrups, aqueous or oil suspensions and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions, include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.
Where the processes for the preparation of the compounds according to the invention give rise to mixture of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution. The compounds may, for example, be resolved into their components enantiomers by standard techniques, such as the formation of diastereomeric pairs by salt formation with an optically active acid, such as (xe2x88x92)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-l-tartaric acid followed by fractional crystallization and regeneration of the free base. The compounds may also be resolved by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column.
During any of the processes for preparation of the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley and Sons, 1991. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
The method of treating PAR-1 mediated disorders described in the present invention may also be carried out using a pharmaceutical composition comprising any of the compounds as defined herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain between about 0.01 mg to about 100 mg, preferably from about 5 to about 50 mg, of the compound, and may be constituted into any form suitable for the mode of administration selected. Carriers include necessary and inert pharmaceutical excipients, including, but not limited to, binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes, and coatings. Compositions suitable for oral administration include solid forms, such as pills, tablets, caplets, capsules (each including immediate release, timed release and sustained release formulations), granules, and powders, and liquid forms, such as solutions, syrups, elixirs, emulsions, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions and suspensions.
Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders; lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
The liquid forms in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl cellulose and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.
The compound of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residue. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
Compounds of this invention may be administered in any of the foregoing compositions and according to dosage regimens established in the art whenever treatment of PAR-1 mediated disorders is required.
The daily dosage of the products may be varied over a wide range from about 0.01 mg to about 1,000 mg per adult human per day. For oral administration, the compositions are preferably provided in the form of tablets containing about 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.01 mg/kg to about 100 mg/kg of body weight per day. Preferably, the range is from about 0.03 mg/kg to about 10 mg/kg of body weight per day. The compounds may be administered on a regimen of about 1 time per day to about 4 times per day.
Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular compound used, the mode of administration, the strength of the preparation, the mode of administration, and the advancement of the disease condition. In addition, factors associated with the particular patient being treated, including patient age, weight, diet and time of administration, will result in the need to adjust dosages.
The compounds of the present invention are thrombin receptor (PAR-1) antagonists. The compounds interrupt platelet activation induced by thrombin""s proteolytic cleavage of its platelet surface receptor, and thereby inhibit platelet aggregation. Such compounds are, therefore, useful in treating platelet-mediated thrombotic disorders (e.g., arterial and venous thrombosis, acute myocardial infarction, reocclusion following thrombolytic therapy and angioplasty, and a variety of vaso-occlusive disorders) and other PAR-1 mediated disorders.
In Vitro Thrombin Receptor Binding Assay
CHRF membranes (Jones, Biochim. Biophys. Acta 1992, 1136, 272) are thawed from xe2x88x9270xc2x0 C., centrifuged at maximum speed for 5 min, washed twice with binding buffer (50 mM HEPES containing 5 mM MgCl2 and 0.1% BSA), and re-suspended in binding buffer (25 xcexcg/100 mL). 100 xcexcL of membranes are added to the 24-Wallac plates and delivered to the Tomtech apparatus. In a typical experiment, 6 xcexcL of samples (from a 125 xcexcg/mL intermediary plate, 20% DMSO) and 44 xcexcL buffer are delivered to the plates (final. conc. of compounds is 3.7 xcexcg/mL, 0.6% DMSO). Similarly, 6 xcexcL 20% DMSO and 44 xcexcL buffer are delivered to both column 1 (NSB) and column 12 (TB). 10 xcexcL Ser-pFPhe-Har-Leu-Har-Lys-Tyr-NH2, SEQ ID NO 1, (721-40; 500 xcexcM in deionized water) is added to column 1. 50 xcexcL tritiated 721-40 (specific activity 46 Ci/mmol) is added to all the wells. The plates are mixed well for 20 seconds, incubated for 30 min, and then harvested with 10 mM HEPES/138 mM NaCl using the Skatron harvester. The filters (GF/C Brandel FPXLR 296) are presoaked 3 h in 0.5% polyethylenimine in HEPES/0.1M N-acetylglucosamine) are set in saran wrap and dried for 3 min in the microwave, and placed in sample bags (Wallac 1450-432). 4.5 mL scintillation fluid (Wallac, Betaplate Scint 1205-440) is added. The bags are sealed, placed in filter cassettes (Wallac 1450-104), and analyzed on the microbeta counter.
In Vitro Inhibition of Thrombin-induced Gel-filtered Platelet Aggregation Assay
The percentage of platelet aggregation is calculated as an increase in light transmission of compound-treated platelet concentrate vs. control-treated platelet concentrate. Human blood is obtained from drug free, normal donors in tubes containing 0.13M sodium citrate. Platelet rich plasma (PRP) is collected by centrifugation of whole blood at 200xc3x97g for 10 min at 25xc2x0 C. The PRP (5 mL) is gel filtered through Sepharose 2B (bed volume 50 mL), and the platelet count is adjusted to 2xc3x97107 platelets per sample. The following constituents are added to a siliconized cuvette: concentrated platelet filtrate and Tyrode""s buffer (0.14M NaCl, 0.0027M KCl, 0.012M NaHCO3, 0.76 mM Na2HPO4, 0.0055M glucose, 2 mg/mL BSA and 5.0 mM HEPES @ pH 7.4) in an amount equal to 350 xcexcL, 50 xcexcL of 20 mM calcium and 50 xcexcL of the test compound. Aggregation is monitored in a BIODATA aggregometer for the 3 min following the addition of agonist (thrombin 50 xcexcL of 1 unit/mL).
Tables 1 and 2 show the biological activity of the compounds of the present invention. Tables 1 and 2 contain IC50 values (xcexcM) of the compounds against platelet aggregation stimulated by thrombin and IC50 values (xcexcM) in a thrombin receptor (PAR-1) binding assay.